The phytochromes comprise a family of biliprotein photoreceptors which enable plants to adapt to their prevailing light environment (Kendrick and Kronenberg (1994) Kendrick, Pp. 828 in Photomorphogenesis in Plants, Dordrecht, The Netherlands: Kluwer Academic Publishers). All phytochromes possess the ability to efficiently photointerconvert between red light absorbing Pr and far red light absorbing Pfr forms, a property conferred by covalent association of a linear tetrapyrrole (or bilin) with a large apoprotein. Phytochromes from cyanobacteria, to green algae and higher plants consist of a well conserved N-terminal polypeptide, roughly 390-600 amino acids in length (see FIG. 6), to which the bilin prosthetic group phytochromobilin (P.PHI.B) or phycocyanobilin (PCB) is bound.
The N-terminal domain of the phytochrome apoprotein is sufficient for spontaneous covalent attachment of ethylidene containing linear tetrapyrroles, a process requiring neither cofactors nor additional enzymes (Li et al. (1992) J. Biol. Chem., 267: 19204-19210). In higher plants, P.PHI.B is bound to a conserved cysteine residue within the phytochrome apoprotein via a linkage identical to that found in the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae and cryptomonads. The ability of the phytochrome photoreceptor to self assemble with its bilin prosthetic group contrasts with the phycobiliprotein photoreceptors which require separate enzymes for proper bilin attachment (Glazer (1989) J. Biol. Chem., 264: 1-4). Owing to the efficient photointerconversion between Pr and Pfr forms, phytochromes are poorly fluorescent molecules, unlike the phycobiliproteins which are intensely fluorescent and have been exploited as useful probes (see, e.g., U.S. Pat. Nos. 4,857,474, and 4,520,110).
Fluorescent markers have found uses in molecular biology as labels for nucleic acid probes, antibodies, and other specific binding ligands in the detection of particular target moieties (e.g., particular nucleic acid sequences, receptors, etc.). Labeled binding molecules are used both in vitro and in vivo as diagnostic indicators and as research tools. Consequently there has been considerable interest and research on the development of fluorescent indicators.
Typically biological macromolecules (e.g., proteins or oligonucleotides) are labeled with a fluorescent marker (e.g., fluorescein, rhodamine, umbelliferone, and lanthanide chelates) either directly through a covalent linkage (e.g., a carbon linker), or indirectly whereby the macromolecule is bound to a molecule such as biotin or dioxigenin, which, is subsequently coupled to a fluorescently labeled macromolecular binding moiety (e.g., streptavidin or a labeled monoclonal antibody). Fluorescein and rhodamine are among the most commonly used fluorophore since they are readily available in an activated form for direct coupling to antigens or antibodies. Both fluorescein and rhodamines show good chemical stability and have a proven record in actual use as labels. However, macromolecules labeled with these fluorophores suffer from chemical quenching of fluorescence, and it is difficult to control the labeling of discrete sites within the macromolecule.
These fluorescent labeling systems also suffer the disadvantage that the fluorescent complexes and/or their binding moieties are relatively large, and must be prepared and supplied from an exogenous source because most organisms are not capable of synthesizing these molecules. In addition, these molecules are often toxic to the subject organism.
With only one exception, the Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria (U.S. Pat. No. 5,491,084), the ability to synthesize a fully functional fluorescent macromolecule has been restricted to the host organism in which the protein naturally occurs. Because the nucleic acid encoding GFP can be cloned into a cell and expressed to yield a non-fluorescent protein precursor that spontaneously assembles its own fluorophore, GFP has gained widespread utility as a selectable marker and a probe of cellular events (Cubitt et al. (1995) Trends In Biochem. Sci. 20, 448-455). From many attempts to improve the properties of GFP through genetic engineering, it is clear that there is a finite spectral window within which GFP is useful as a fluorescent marker. The development of additional protein-based fluorescent markers that can be functionally expressed in various cell types by standard genetic engineering techniques with an extended fluorescence wavelength range, and a variety of useful biochemical properties is desirable.
A recent development in the field of fluorescent labeling has been the use of phycobiliprotein conjugates. Phycobiliproteins are a class of highly fluorescent proteins that form a part of the light-harvesting system in the photosynthetic apparatus of bluegreen bacteria and of two groups of eukaryotic algae, red algae and the cryptomonads. A particularly useful variation of their use comprises preparation of a phycobiliprotein tandem conjugate with a large Stokes shift. An example of such a conjugate is the covalent attachment of the phycobiliproteins, phycoerythrin and allophycocyanin. The resulting tandem conjugate has a large Stokes shift with an emission maximum at 660 nm and an excitation waveband that starts at about 440 nm. However, production of such tandem complexes requires human intervention in the formation of a covalent or other chemical bond between the two components, therefore increasing the complexity of the production of the final conjugate.
Despite these advances, the art fails to provide fluorescent markers that can be easily produced and readily engineered to provide strong fluorescent signals over a wide range of wavelengths. The present invention addresses these and other needs.